DESCRIPTION: In all animal cells, the presence of impermeant anions in the intracellular compartment creates Gibbs-Donnan forces which tend to produce colloid-osmotic swelling. Cells evolved mechanisms for maintaining their volume constant in isosmotic media, thus preventing osmotic swelling and lysis. Failure of these mechanisms underlies the pathophysiology of osmotic cell damage. Neurons face unique and potentially severe challenges for cell volume homeostasis. Net accumulation or depletion activity, or in pathological conditions such as ischemia, trauma, seizures or metabolic disorders. In spite of its clinical and therapeutic implications, knowledge on physiology and pathology of cell volume homeostasis in neuronal cells is scarce. This is a proposal to study the cellular and molecular mechanisms by which neuronal cells regulate and maintain their water volume upon changes in extracellular or intracellular osmolality elicited by external stimuli. We aim to ascertain the role of ionized calcium (Ca2+) as an intracellular signal coupling external stimuli with effectors controlling cell volume decrease in cells swollen in hyposmotic media, and characterize pharmacologically the membrane transport systems effecting this response. In isosmotic medium, some hormones, peptides, and neurotransmitters, or inhibition of the Na+/K+ pump, produce neuronal shrinkage which seems to be mediated by an increase in [Ca2+]. We will also study the role of Ca2+ as mediator of these responses and the mechanisms leading to restoration of cell volume in isosmotic media. The consequences of inhibition of the Na+/K+ pump on osmotic balance and cell survival following acute Na+ loads, like those occurring under pathological conditions, will be determined. These studies will be conducted using, as in vitro models, murine neuronal cell lines and land snail neurons under strictly defined conditions. Changes in water volume and intracellular ion activities will be measured in single cells using fluorescent probes with newly developed optical methods. Transmembrane current will be studied with voltage clamp techniques.